Notch3 and DeltaB maintain Müller glia quiescence and act as negative regulators of regeneration in the light-damaged zebrafish retina.

Damage to the zebrafish retina stimulates resident Muller glia to reprogram, reenter the cell cycle, divide asymmetrically, and produce neuronal progenitor cells that amplify and differentiate into the lost neurons. The transition from quiescent to proliferative Muller glia involves both positive and negative regulators. We previously demonstrated that the Notch signaling pathway represses retinal regeneration by maintaining Muller glia quiescence in zebrafish. Here we examine which Notch receptor is necessary to maintain quiescence. Quantitative RT-PCR and RNA-Seq analyses reveal that notch3 is expressed in the undamaged retina and is downregulated in response to light damage. Additionally, Notch3 protein is expressed in quiescent Muller glia of the undamaged retina, is downregulated as Muller glia proliferate, and is reestablished in the Muller glia. Knockdown of Notch3 is sufficient to induce Muller glia proliferation in undamaged retinas and enhances proliferation during light damage. Alternatively, knockdown of Notch1a, Notch1b, or Notch2 decreases the number of proliferating cells during light damage, suggesting that Notch signaling is also required for proliferation during retinal regeneration. We also knockdown the zebrafish Delta and Delta-like proteins, ligands for the Notch receptors, and find that the deltaB morphant possesses an increased number of proliferating cells in the light-damaged retina. As with Notch3, knockdown of DeltaB is sufficient to induce Muller glia proliferation in the absence of light damage. Taken together, the negative regulation of Muller glia proliferation in zebrafish retinal regeneration is mediated by Notch3 and DeltaB.